Assessment of a High-Resolution HPLC System to Quantify HbE for α⁰-Thalassemia Diagnosis in HbE Heterozygotes

HbE and α⁰-thalassemia are highly prevalent in Southeast Asia and co-inherited in asymptomatic carriers. α⁰-Thalassemia screening in HbE heterozygotes relies on HbE quantification. We evaluated the performance of a high-resolution HPLC system in quantifying HbE and HbA₂ to distinguish HbE heterozygotes with and without α⁰-thalassemia.

HbE heterozygotes (N=650) presenting EA patterns on capillary electrophoresis (CE) were analyzed using the Premier Resolution (PR) HPLC system (Trinity Biotech). HbA₂ and HbE levels were quantified, Pearson correlations with CE assessed, and diagnostic cutoffs determined. A prospective cohort of HbE heterozygotes (N=884) was used for validation. α⁰-Thalassemia genotyping was performed using gap-PCR.

PR-HPLC confirmed HbE heterozygosity and successfully separated HbA₂ from HbE. The HbA₂, HbE, and combined HbA₂+HbE levels were consistently higher than but correlated with CE values (HbA₂: bias=1.213, r=0.686; HbE: bias=0.286, r=0.960; combined: bias=1.502, r=0.952). HbE and combined levels were substantially lower in α⁰-thalassemia carriers (14.65%±2.2% vs. 19.38%±2.5%); HbA₂ levels did not significantly differ. HbE cutoff <19% resulted in an area under the curve (AUC) of 0.975, with 100% sensitivity and 91.9% specificity. HbE+HbA₂ <24.0% yielded an AUC of 0.970, with 100% sensitivity and 89.7% specificity. Validation experiments supported the robust performance for HbE detection alone (100% sensitivity, 93.6% specificity, and 94.5% accuracy).

PR-HPLC can quantify HbE precisely, potentially aiding in robust α⁰-thalassemia diagnosis and screening without HbA₂ assessment.